Mass spectrometric studies on the peptide integrity of substance P and related human tachykinins in human biofluids.

The neurokinin-1 receptor plays a profound role in inflammatory processes and is involved in immune cell differentiation, cytokine release, and mast cell activation. Due to their similar peptide structures, the neurokinin-1 receptor does not discriminate between the endogenous ligands substance P (SP) and human hemokinin-1 (hHK-1), which both demonstrate biological receptor affinity. In addition, due to cross-reactivity, the current bioanalytical method of choice-immunoassays-also displays limitations in differentiating between these peptides. Thus, a recently developed mass spectrometric assay was utilized for the selective quantification of SP and hHK-1 in various biofluids and tissue. By applying the sample processing protocols developed, SP was quantified in porcine brain tissue (4.49 ± 0.53 nM), human saliva (113.3 ± 67.0 pM), and human seminal fluid (0.52 ± 0.15 nM) by mass spectrometric analysis. As previously reported, neither SP nor hHK-1 could be detected in human plasma by mass spectrometry. Comparison with analysis using a commercial immunoassay of the same plasma sample revealed SP like-immunoreactivity concentrations of 37.1-178.0 pM. The previously reported carboxylic acid of SP, whose identity was confirmed by high-resolution mass spectrometric analysis, did not show cross-reactivity in the applied immunoassay and did not contribute to SP-like immunoreactivity results. Subsequent compound discovery of the immunocaptured substance indicated the presence of a precursor of SP as possible cross-reactor in human plasma samples. The found cross-reactivity might be the cause for the high variance of SP plasma levels in former determinations.

Crystal structures of the human neurokinin 1 receptor in complex with clinically used antagonists.

Neurokinins (or tachykinins) are peptides that modulate a wide variety of human physiology through the neurokinin G protein-coupled receptor family, implicated in a diverse array of pathological processes. Here we report high-resolution crystal structures of the human NK1 receptor (NK1R) bound to two small-molecule antagonist therapeutics - aprepitant and netupitant and the progenitor antagonist CP-99,994. The structures reveal the detailed interactions between clinically approved antagonists and NK1R, which induce a distinct receptor conformation resulting in an interhelical hydrogen-bond network that cross-links the extracellular ends of helices V and VI. Furthermore, the high-resolution details of NK1R bound to netupitant establish a structural rationale for the lack of basal activity in NK1R. Taken together, these co-structures provide a comprehensive structural basis of NK1R antagonism and will facilitate the design of new therapeutics targeting the neurokinin receptor family.

Cloning of a C-terminally truncated NK-1 receptor from guinea-pig nervous system.

In order to examine the possibility that some actions of substance P may be mediated by a variant of the neurokinin-1 (NK-1) receptor, we isolated and sequenced the cDNA encoding a truncated NK-1 receptor from guinea-pig celiac ganglion and brain mRNA by two-step RT-PCR based on the 3'RACE method. The truncated NK-1 receptor sequence corresponded to a splice variant missing the final exon 5, and encoded a 311-amino acid protein that was truncated just after transmembrane domain 7, in an identical position to a truncated variant of the human NK-1 receptor. Thus, the truncated NK-1 receptor lacked the intracellular C-terminus sequence required for the phosphorylation and internalisation of the full-length NK-1 receptor. Using a sensitive one-step semi-quantitative RT-PCR assay, we detected mRNA for both the full length and truncated NK-1 receptors throughout the brain, spinal cord, sensory and autonomic ganglia, and viscera. Truncated NK-1 receptor mRNA was present in lower quantities than mRNA for the full-length NK-1R in all tissues. Highest levels of mRNA for the truncated NK-1 receptor were detected in coeliac ganglion, spinal cord, basal ganglia and hypothalamus. An antiserum to the N-terminus of the NK-1 receptor labelled dendrites of coeliac ganglion neurons that were not labelled with antisera to the C-terminus of the full length NK-1 receptor. These results show that a C-terminally truncated variant of the NK-1 receptor is likely to be widespread in central and peripheral nervous tissue. We predict that this receptor will mediate actions of substance P on neurons where immunohistochemical evidence for a full-length NK-1 receptor is lacking.

Neurons in the dorsal column white matter of the spinal cord: complex neuropil in an unexpected location.

It is common to think of gray matter as the site of integration in neural circuits and white matter as the wires that connect different groups of neurons. The dorsal column (DC) white matter, for example, is the spinal cord axonal pathway through which a topographic map of the body is conveyed to the somatosensory cortex. We now describe a network of neurons located along the midline of the DCs. The neurons are present in several mammals, including primates and birds, and have a profuse dendritic arbor that expresses both the neuron-specific marker, microtubule-associated protein-2, and the neurokinin-1 receptor, a target of the neuropeptide, substance P. Electron microscopy and double immunostaining for synaptophysin and a marker of gamma-aminobutyric acid-ergic terminals documented a rich synaptic input to these neurons. Finally, injection of a gamma-aminobutyric acid type A receptor antagonist or of substance P into the cerebrospinal fluid of the rat spinal cord induced Fos expression and internalization of the neurokinin-1 receptor in these neurons, respectively, indicating that the DC neurons are under tonic inhibitory control and can respond to neurotransmitters that circulate in the cerebrospinal fluid.

Substance P (neurokinin-1) receptor is a marker of human mucosal but not peripheral mononuclear cells: molecular quantitation and localization.

Reciprocal communication between the immune system and the neuroendocrine system is mediated via a common chemical language of shared ligands and receptors. The neuropeptide substance P (SP) has been implicated as a mediator of immunomodulation. The evidence for substance P receptors on human lymphocytes is, however, controversial. The aims of the present study are to investigate substance P receptor (SPR) expression in human peripheral and mucosal mononuclear cells and to identify cellular sites of expression in human colonic mucosa. Using reverse-transcriptase PCR, we demonstrate that PBMC isolations are negative for SPR mRNA expression, whereas lamina propria mononuclear cell (LPMC) isolations express on average eight SPR mRNA transcripts per cell. In situ hybridization performed on surgically resected colonic tissue confirms the expression of SPR mRNA in LPMC in vivo. SPR mRNA signal was detected in LPMC, lymphoid follicles, and epithelium. The complementary technique of immunohistochemistry gave a similar distribution of SPR expression that colocalized with CD45 immunoreactivity. Dual-fluorochrome flow cytometry revealed SPR expression by CD4, CD45RO, CD45RA, CD8, CD19, and CD14 LPMC subsets, but not PBMC. Our findings suggest that SPR expression is distinctive of human colonic mucosal mononuclear cells and support a direct role for SP in mucosal immunomodulation.

Autoradiographic localization of neurotransmitter binding sites in the hypoglossal and motor trigeminal nuclei of the rat.

The hypoglossal and motor trigeminal nuclei contain somatic motoneurons innervating the tongue, jaw, and palate. These two cranial motor nuclei are myotopically organized and contain neurotransmitter binding sites for thyrotropin-releasing hormone, substance P, and serotonin. Quantitative autoradiography was used to localize thyrotropin-releasing hormone, substance P, and serotonin-1A and serotonin-1B binding sites in the hypoglossal and motor trigeminal nuclei and to relate the relative distributions of these binding sites to the myotopic organizations of the two nuclei. In the hypoglossal nucleus, high-to-moderate concentrations of all four binding sites were present in the dorsal and ventromedial subnuclei, whereas low concentrations were noted in the ventrolateral subnucleus. In the motor trigeminal nucleus, high concentrations of serotonin-1B, moderate densities of thyrotropin-releasing hormone, and low levels of substance P and serotonin-1A binding sites were present in both the ventromedial and dorsolateral subnuclei. These observations demonstrate that neurotransmitter binding sites in the hypoglossal and motor trigeminal nuclei are heterogeneously localized and that their distributions correspond to the previously described myotopic organizations of each nucleus.

Purification and reconstitution of a recombinant human neurokinin-1 receptor.

Recombinant human neurokinin-1 receptors expressed in insect cells have been purified to near homogeneity by sequential metal-chelating chromatography and gel filtration chromatography. The purified receptor consists of a single polypeptide with an apparent molecular weight of 50 kD as revealed by SDS gel electrophoresis, and exhibits a specific activity of 19 nmol of L-703,606 bound per mg of protein. Immunoblot experiments further confirm the identity of the stained protein band. The purified receptor binds the antagonist L-703,606 with an affinity similar to that of native human neurokinin-1 receptor, and binds the agonist substance P with an affinity similar to that of the low affinity state of uncoupled native receptor. The purified receptor can be reconstituted with membranes from uninfected insect cells, and the reconstitution results in an increased affinity for substance P, consistent with the reappearance of the high affinity state of the receptor for agonist in the presence of endogenous G proteins. These data indicate that the purified neurokinin-1 receptor is functional with respect to agonist and antagonist binding and G protein coupling.

Chemical cross-linking of the substance P (NK-1) receptor to the alpha subunits of the G proteins Gq and G11.

We have previously shown that the high-affinity binding of substance P (SP) to its receptor is dependent on an interaction with a PTX-insensitive G protein. This G protein couples SP receptor activation to stimulation of its effector, phospholipase C. In this study, we combined photoaffinity labeling, chemical cross-linking techniques, and immunological characterization using sequence-specific antibody probes to identify G proteins that couple to the SP receptor. First we covalently labeled the SP receptor present on rat submaxillary gland membranes with a radioiodinated photoreactive derivative of SP, p-benzoyl-L-phenylalanine(8)-substance P (125I-[Bpa8]SP). Photoincorporation of this SP derivative was susceptible to guanine nucleotide inhibition, indicating that the receptor was coupled to its G protein during labeling. We then used a chemical cross-linking agent to covalently link the photoaffinity labeled SP receptor and its associated G protein. Cross-linking generated a 96 kDa product, formation of which was prevented by the addition of a guanine nucleotide, but not an adenine nucleotide, following photolabeling, but prior to cross-linking. Furthermore, the 96 kDa cross-linked complex was absent in membranes which had been depleted of G proteins by treatment with alkaline buffer prior to addition of the cross-linking agent. Reductive cleavage of the cross-link in the isolated 96 kDa complex yields two products: the 53 kDa SP receptor and a 42 kDa protein identified by immunoblot analysis as either G alpha q or G alpha 11. Antisera against a common sequence within G alpha s, G alpha i, and G alpha o showed no immunoreactivity to the complex or its cleavage products. These results provide the first direct evidence of specific interaction between photoaffinity labeled SP receptor and the alpha subunits of Gq and G11, members of a family of G proteins known to be associated with pertussis toxin-insensitive phospholipase C activation.